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LINEAR GRADIENT GELS

For Lamin gels, pour from top down by attaching tubing to top of the gels plates. ****Heavy mix goes in the right side of the gradient mixer, Light mix goes on left side (no plunger is needed)****

Gradient: Acrylamide 7.5% to 15% Sucrose 5% to 15%

Light Heavy Resolving Gel: 4X Lower Tris Base 4.8ml 4.8ml (pH= 8.8)

40% Acrylamide/ Bis 3.75 7.5 (29:1)

66% Sucrose (1.93M) 1.5 4.6

dH2O 9.95 3.1

10% SDS 200ul 200ul

Before adding the polymerizing agents to the heavy and light mixes, be sure that the tubing is placed in the correct place on the gel plates, that the gradient mixer valves are in the upright position, and that the pump is on the correct setting (needs to be at speed 3)

Polymerizing Agents: TEMED 5ul 5ul

10% Ammonium persulfate 50ul 50ul

Turn the pump on by moving the switch to the "forward" position.

After the gel has been poured, quickly turn the pump off and remove the tubing from the top of the gel plates. Place the tubing in the water collecting bottle next to the pump. Fill the gradient mixer with water and turn the pump back on to prevent the tubing and the pump from clogging with the polymerizing solution.

Overlay the gel with a thin layer of butanol which has been saturated in the Lower Tris Base Buffer. Let the gel polymerize for at least 2hrs. Prepare the stacking gel.

Be sure to pour off the butanol from the top of the polymerized gel and rinse with dH2O. Blot the gel with a paper towel before pouring the stacker. The stacker can be "poured" by using a Pasteur pipet. After pouring, quickly wedge the comb between the two plates. Let polymerize for at least a half hour.

Stacker for: 1 gel 2 gels 3 gels

4X Upper Tris Base 1.88ml 3.75ml 4.63ml (pH= 6.8)

40% Acrylamide/ Bis .95 1.9 2.85 (29:1)

50% Glycerol .75 1.5 2.25

dH2O 3.9 7.8 11.7

10% SDS 75ul 150ul 225ul

Polymerizing Agents: TEMED 5ul 10ul 15ul

10% Ammonium persulfate 50ul 100ul 150ul

Once the gel and stacker have both polymerized, remove the clamps and gasket. Rinse the side of the plate that will be against the gel box with 95% EtOH until dry. Also, be sure that the gel box is rinsed with 95% EtOH until clean and dry. Fix the gel plates to the gel box with clamps (the comb should be facing the box). Use agarose to seal the plates to the box. Fill the top and bottom chambers with SDS running buffer, then remove the comb slowly. Wash the wells and the bottom of the gel with running buffer using a syringe. Load samples and run the gel overnight at 9 mAmp constant current.